The invention relates to a process for producing monoclonal antibodies to bluetongue virus antigen and to the hybrid cell lines that secrete these monospecific antibodies.
Bluetongue (BT) is an infectious, non-contagious, insect-transmitted virus disease that affects sheep, cattle, and other species of ruminant animals. Bluetongue virus (BTV) is classified as an orbivirus and is antigenically related to epizootic hemorrhagic disease virus (EHDV), also an orbivirus. Presently, 23 serotypes of BTV have been identified in the world and five serotypes are known to be present in the United States.
BTV infection in sheep is usually acute with a mortality rate in infected sheep of about 20-50%. Clinical symptoms of illness due to BTV are less apparent in cattle than in sheep and over 90% of BTVinfected cattle have mild or undetectable clinical signs of infection. However, infected cattle are important in the epizootiology of BT because cattle are often reservoirs of the virus. In addition, although BTV causes more direct losses in sheep production, significant economic losses result from BTV infections in cattle due to embargoes and restriction of export markets for cattle.
Proper treatment for BTV depends on accurate diagnosis of the disease. Problems in diagnosing BTV result, however, becuse EHDV, a closely related orbivirus, also infects cattle and produces a similar clinical syndrome. Because the two diseases are so similar it is necessary to isolate the viral agent and correctly identify it in order to facilitate an accurate diagnosis. One method that has been used to differentiate between BTV and EHDV is the indirect fluorescent antibody test (IFAT), which uses hyperimmune rabbit serum taken from BTV- or EHDV-inoculated rabbits to visualize viral antigen in virus-infected cell cultures. Because BTV and EHDV "share" antigenic sites on some of the polypeptides, even the most specific rabbit serum will contain antibodies that cross-react with viral antigens associated with both BTV and EHDV. Heretofore it has been necessary to dilute the cross-reacting antibody to a point where the serum became specific only for the homologous virus. However, there is always a possibility of incorrectly identifying BTV with this system. What has been needed to improve the capability of the diagnostic laboratory to differentiate between BTV and EHDV is an antibody that will identify BTV without cross-reacting with the antigenically related EHDV. In addition, because more than one serotype of BTV can be present in an infected animal and/or an infection outbreak area, it is highly desirable that such a BTV-specific antibody be group-specific, that is, would recognize any of the serotypes of BTV that might be present, rather than only be specific to one serotype. It is also desirable to have a method of visualizing BT viral-specific antigens in cell cultures.
The production of monoclonal antibodies by the fusion of spleen cells from immunized mice and myeloma cells grown in continuous culture, has been described previously, e.g., Kohler et al. in Nature, Vol. 256, pp. 495-497 (1975), Kohler et al. in European Journal of Immunology, Vol. 6, pp. 511-519 (1976), Galfre et al. in Nature, Vol. 266, pp. 550-552 (1977), and in the text Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, R. Kennett, T. McKearn, K. Bechtol, Eds., Plenum Press, N.Y. and London, (1980). Techniques for the chemical selection of hybridomas arising from such a fusion, and subsequent isolation of single antibody secreting cell clones for the production of the monoclonal antibodies are also known. However, no cell lines have been produced capable of secreting monoclonal antibodies which are group-specific to BTV, rather than serotype-specific, which do not give false positive reactions to obiviruses such as EHDV, and which have been shown to be useful for identification of BTV antigens using simple tests such as immunofluorescence.
It should be noted that because of the unpredictable nature of hybrid cell preparation one cannot extrapolate from one antigen or cell system to another.